Compositions and methods for detection and treatment of proliferative abnormalities associated with overexpression of human transketolase like-1 gene

ABSTRACT

Methods for in vitro diagnosis of carcinoma are disclosed. In one aspect the invention relates to methods such as detecting the level of transketolase like-1 polypeptide, which are especially useful for the detection of tumors and their precursory stages based on the detection of overexpression of human transketolase like-1 gene in biological samples. In another aspect the invention relates to methods for treatment of disorders associated with the overexpression of human transketolase like-1 gene. Methods for treatment may include gene therapeutic approaches as well as methods for inhibiting or reducing the activity of transketolase like-1 polypeptides.

The present invention relates to methods for treatment and diagnosis ofdisorders associated with abnormally proliferating cells. In one aspectthe invention relates to methods, which are especially useful for thedetection of tumors and their precursory stages based on the detectionof overexpression of human transketolase like-1 gene in biologicalsamples. In another aspect the invention relates to methods fortreatment of disorders associated with the overexpression of humantransketolase like-1 gene. Methods for treatment may include genetherapeutic approaches as well as methods for inhibiting or reducing theactivity of transketolase like-1 polypeptides.

Despite significant scientific and medical research efforts, neoplasticdiseases still remain a major cause of human mortality. For example eachyear more than 340,000 persons in Germany develop cancer and more than210,000 die from their disease. Epithelial tumors represent the majorityof cancer: Lung cancer is the leading cause of cancer deaths in males,and breast cancer is the leading cause in females. The second leadingcause of cancer deaths for both sexes is colorectal cancer (Becker, N.and Wahrendorf, J., (1997) Atlas of Cancer Mortality in the Federalrepublic of Germany 1981-1990, Springer-Verlag, Berlin, Heidelberg).

One major reason for this unsatisfying situation is, that mostneoplastic diseases are diagnosed at relatively late stages, whenisolated tumor cells or small tumor cell aggregates were alreadyreleased from the primary tumor and distributed in the whole organism ofthe host and might have eventually already caused occult or frankmetastatic disease. Early cancers and in particular precancers usuallydo not cause any symptoms and are not realized by the respectivepatients.

To overcome this, more research efforts and clinical programs arerequired to improve cancer early detection technologies, as well as todevelop true preventive or therapeutic vaccination strategies toimmunize patients either before a defined cancer emerged or afterresection of a cancer or its precursors to prevent survival ofdisseminated isolated cancer cells (DTCs) which might have been releasedfrom the neoplasm either before or during primary surgical intervention.

For few cancers, in particular cancer of the uterine cervix efficientcancer early detection programs could be established. The subsequentreduction of mortality rates associated with these specific neoplasmsconvincingly demonstrated the high effectiveness of the early detectionprograms.

To summarize, unfortunately, the diagnostic methods used so far arerelatively insensitive and take the risk to yield false-positive resultsdue to lack of specificity. Moreover, by using the current diagnosticmethods any conclusions as regards the grade of malignancy, theprogression of the tumor and its potential for metastasising cannot beprecisely predicted.

Thus, the use of reliable diagnostic molecular markers would be highlybeneficial for an understanding of the molecular basis of epithelialtumors, e.g. colon tumors, for distinguishing benign from malignanttissue and for grading and staging carcinomas, particularly for patientswith metastasising cancer having a very bad prognosis. It can beexpected that such markers are also useful for the development of noveltherapeutic avenues for cancer treatment.

The understanding of the molecular events underlying the transition of anormal cell into a tumor cell of different grades of aggressiveness andthe availability of appropriate experimental systems to select forcancer-associated genes are absolute prerequisites for theidentification of such novel diagnostic markers and therapeutic drugtargets.

It is commonly accepted that tumorigenesis represents a complexmultistage process in which genetic changes and environmental factorsare thought to deregulate the cellular processes that control cellproliferation and differentiation. This multistep process is wellillustrated for example by colorectal cancers, which typically developover decades and appear to require multiple genetic events forcompletion (for review Kinzler and Vogelstein, 1996, Cell 87, 159-170).Both inheritance of altered genes (resulting in a marked predisposition)and genomic instability (caused by genotoxic agents from theenvironment) resulting in additional somatic mutations contribute tothis process. Clearly, the list of decisive players causally involved intumor formation is far from being complete and will obviously varydepending on the type of tumor.

Thus, the technical problem underlying the present invention is toprovide means for diagnosis and therapy of epithelial tumors, whichovercome the disadvantages of the presently available diagnostic andtherapeutic methods.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

The present invention is based on the inventors findings, that humantransketolase like-1 gene as given in SEQ. ID. 1 (cf. TKT-L1, TKR:NM_(—)012253; Accession number: X91817) is highly overexpressed intissue of colon carcinoma, pancreatic carcinoma, lung cancer and gastriccancer compared to the level found in respective normal control tissue.This is especially valuable for diagnostic purposes, as transketolaseenzyme is not comparably overexpressed in tumour tissue.

Thus a method for diagnosis of tumors can be based on the detection ofoverexpression of transketolase like-1 gene products in biologicalsamples. According to the detected presence or absence and/or level oftransketolase like-1 gene products it is possible to predict the diseasecourse, to assess prognosis and to tailor adequate therapy for patients.

Furthermore the invention enables for therapeutic methods applicable todisorders associated with the overexpression of transketolase like-1gene products. On the one hand the invention provides methods usingtransketolase like-1 nucleic acids or polypeptides for the therapy ofdisorders. On the other hand the invention provides for methods based onthe reduction of the enzymatic activity of transketolase like-1 genepolypeptides. Thus it is one aspect of the invention to provide a methodfor rational tumor management based on the detection of transketolaselike-1 gene products in patient samples and the tailoring of a therapycorrelated to the detected overexpression of said gene products.

Finally the present invention relates to diagnostic and research kitsand to pharmaceutical compositions useful for performing the methodsdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Detection of the overexpression of transketolase like-1 gene byRT-PCR in colon carcinomas; the diagram shows the induction ofexpression of the transketolase like-1 gene in tissue of colon carcinomain comparison to control tissue.

FIG. 2: Detection of the overexpression of transketolase like-1 gene byRT-PCR in lung adenocarcinomas; the diagram shows the induction ofexpression of the transketolase like-1 gene in tissue of lungadenocarcinoma in comparison to control tissue.

FIG. 3: Detection of the overexpression of transketolase like-1 gene byRT-PCR in carcinomas of the stomach; the diagram shows the induction ofexpression of the transketolase like-1 gene in tissue of carcinomas ofthe stomach in comparison to control tissue.

FIG. 4: Detection of the overexpression of transketolase by RT-PCR incolon carcinomas; the diagram shows the induction of expression oftransketolase in tissue of colon carcinoma in comparison to controltissue.

FIG. 5: Detection of the overexpression of transketolase by RT-PCR inlung adenocarcinomas; the diagram shows the induction of expression oftransketolase in tissue of lung adenocarcinoma in comparison to controltissue.

FIG. 6: Detection of the overexpression of transketolase by RT-PCR incarcinomas of the stomach; the diagram shows the induction of expressionof transketolase in tissue of carcinoma of the stomach in comparison tocontrol tissue.

FIG. 7: DNA and amino acid sequence of tktl1; the part of the proteinbeing used for the immunization for antibody generation is given in boldletters; additionally peptides are underlined which were used forpeptide immunization for antibody generation.

FIG. 8: Immunohistochemical analysis of gastric carcinoma (B) andcorresponding normal tissue (A) employing a primary antibody directedagainst tktl1. In the carcinoma of patient 1666 a strong overexpressionof the tktl1 protein is detectable.

FIG. 9: Immunohistochemical analysis of gastric carcinoma patient 1682(A) and 1697 (B) employing a primary antibody directed against tktl1. Inthe carcinoma of patient 1682 a strong overexpression of the tktl1protein is detectable in the nucleus and the cytoplasm. In the carcinomaof patient 1697 a very strong overexpression of the tktl1 protein isdetectable in the nucleus and the cytoplasm.

FIG. 10: Immunohistochemical analysis of gastric carcinoma patient 1699employing a primary antibody directed against tktl1. FIG. A shows acarcinoma with an area of normal tissue. Whereas in the normal tissue alow expression of tktl1 is detectable a strong overexpression of tktl1is present in the tumor cells of the carcinoma. A magnification of theborder between normal and tumor tissue is shown in B. A tumor specificgranular staining pattern is detectable.

FIG. 11: Immunohistochemical analysis of gastric carcinoma patient 1698employing a primary antibody directed against tktl1. In FIG. A a strongoverexpression of the tktl1 protein is detectable in the nucleus and thecytoplasm of gastric tumor cells. A low or absent expression isdetectable in surrounding fibroblasts. A magnification of an area ofcarcinoma cells with surrounding connective tissue is shown in B. Atumor specific granular staining pattern is detectable.

The present invention provides methods for detection and treatment ofdisorders characterized by abnormal cell proliferation, such as e.g.cancers.

It is a first aspect of the present invention to provide a method forthe detection of disorders characterized by abnormal cell proliferation,such as e.g. cancers based on the determination of the presence orabsence and/or the level of expression of human transketolase like-1gene as given in SEQ. ID. 1 (cf. TKT-L1, TKR: NM_(—)012253; Accessionnumber: X91817) in biological samples.

It is a second aspect of the present invention to provide a method fortreatment of disorders characterized by abnormal cell proliferation,such as e.g. cancers using human transketolase like-1 gene products astherapeutically active agents.

A third aspect of the present invention is a research or diagnostic testkit for performing the reactions involved in the detection of thepresence or absence and/or the level of overexpression of humantransketolase like-1 gene.

A fourth aspect of the present invention relates to pharmaceuticalcompositions applicable in the treatment of disorders according to thepresent invention.

Transketolase like-1 gene products as used in the context of the presentinvention may comprise polypeptides and nucleic acids encoded by thetransketolase like-1 gene.

The polypeptides and polynucleotides used for performing the methodaccording to the present invention are isolated. This means that themolecules are removed from their original environment. Naturallyoccurring proteins are isolated if they are separated from some or allof the materials, which coexist in the natural environment.Polynucleotides are isolated for example if they are cloned intovectors.

Human transketolase like-1 nucleic acid molecules used for performing amethod according to the present invention may comprise polynucleotidesor fragments thereof. Preferred polynucleotides may comprise at least 20consecutive nucleotides, preferably at least 30 consecutive nucleotidesand more preferably at least 45 consecutive nucleotides, that areidentical, share sequence homology or encode for identical, orhomologous polypeptides, compared to the wild type transketolase like-1polypeptides, but do not encode other transketolase like polypeptides ortransketolases. The nucleic acids according to the present invention mayalso be complementary or reverse complementary to any of saidpolynucleotides. Polynucleotides may for example include single-stranded(sense or antisense) or double-stranded molecules, and may be DNA(genomic, cDNA or synthetic) or RNA. RNA molecules comprise as wellhnRNA (containing introns) as mRNA (not containing introns). Accordingto the present invention the polynucleotides may also be linked to anyother molecules, such as support materials or detection markermolecules, and may, but need not, contain additional coding ornon-coding sequences.

The human transketolase like-1 polynucleotides used according to thepresent invention may be native sequences or variants thereof. Thevariants may contain one or more substitutions, additions, deletionsand/or insertions such that the immunogenicity of the encodedpolypeptide is not diminished, relative to the native tumor protein.Variants may for example be allelic variations of the polynucleotides.Allelic variation as used herein is an alternative form of the gene,which may result from at least one mutation in the nucleic acidsequence. Alleles may result in altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one, or many allelic forms. Common mutational changes, which giverise to alleles, are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone or in combination with the others, one or more times in agiven sequence. The variants according to the present invention showpreferably 70%, more preferably at least 80% and most preferably atleast 90% of sequence identity to the native nucleic acid moleculesdisclosed herein. Methods for determination of sequence similarity areknown to those of ordinary skill in the art.

One example for detecting the similarity of sequences may be carried outusing the FastA and/or BlastN bioinformatics software accessible on theHUSAR server of the DKFZ Heidelberg.

Nucleic acids as used in the context of the present invention may be allpolynucleotides, which hybridise to probes specific for thetransketolase like-1 sequences used herein under stringent conditions.Stringent conditions applied for the hybridisation reaction are known tothose of ordinary skill in the art and may be applied as described inSambrook et al. Molecular cloning: A Laboratory Manual, 2nd Edition,1989.

The present invention also comprises polynucleotides, that due to thedegeneracy of the genetic code encode the polypeptides natively encodedby human transketolase like-1 nucleic acids while not showing thepercentage of sequence homology as described above within the nucleicacid sequence. Such nucleic acids may for example arise by changing thecodons present in the disclosed sequences by degenerate codons and sopreparing a synthetic nucleic acid. The preparation of such artificialnucleic acid sequences may be achieved by the methods known to thoseskilled in the art.

The human transketolase like-1 nucleotide sequences used according tothe present invention may be joined to a variety of other nucleic acidsequences using the known recombinant DNA techniques. The sequences mayfor example be cloned into any of a variety of cloning vectors, such asplasmid, phagemids, lambda phage derivatives and cosmids. Furthermorevectors such as expression vectors, replication vectors, probegeneration vectors and sequencing vectors may be joined with thesequences disclosed herein.

Sequences that may be cloned to the nucleic acids according to thepresent invention comprise as well coding sequences as non-codingsequences and regulatory sequences including promoters, enhancers andterminators. The human transketolase like-1 nucleic acid sequencesdisclosed herein might for example be present in combination with othercoding sequences. These sequences may encode for a variety of proteinssuch as enzymes, receptors, antigens, immunogenic fragments or epitopes,binding proteins, etc. The nucleic acid sequences may be joined directlyor may be separated by a stretch of nucleic acids coding for a spacer orlinker region. The nucleic acid sequences may also be separated by astretch of nucleic acids that may be removed after transcription of thesequence. Non-coding sequences, that may be joined to the sequencesdisclosed herein may for example be promoter regions, enhancers, cisregulatory elements, 5′ untranslated regions, terminators etc.

In a preferred embodiment human transketolase like-1 polynucleotides maybe formulated such, that they are able to enter prokaryotic oreukaryotic cells such as mammalian cells and to be expressed in saidcells. Such formulations may be for example useful for therapeuticpurposes. The expression of nucleic acid sequences in target cells maybe achieved by any method known to those skilled in the art. The nucleicacids may for example be joined to elements that are apt to enable theirexpression in a host cell. Such elements may comprise promoters orenhancers, such as CMV-, SV40-, RSV-, metallothionein I- orpolyhedrin-promoters respectively CMV- or SV40-enhancers. Possiblemethods for the expression are for example incorporation of thepolynucleotide into a viral vector including adenovirus,adeno-associated virus, retrovirus, vaccinia virus or pox virus. Viralvectors for the purpose of expression of nucleic acids in mammalian hostcells may comprise pcDNA3, pMSX, pKCR, pEFBOS, cDMB, pCEV4 etc. Thesetechniques are known to those skilled in the art.

Fragments of the human transketolase like-1 sequence used herein maycomprise oligonucleotides such as nucleic acid probes for hybridisationpurposes, primers for amplification reactions or antisense constructsfor use in antisense techniques. Nucleic acid probes according to thepresent invention may be any nucleic acid probe that has a sequence atleast 80% identical to a part of at least 15 consecutive nucleotides ofthe human transketolase like-1 gene nucleic acid sequence or iscomplementary or reverse complementary to such a sequence but does nothybridise to an other transketolase or transketolase like sequence. Thenucleic acid probes according to the present invention are furthermorecharacterized, in that they hybridise under stringent conditions tonucleic acids of the sequence disclosed herein. Primers may be anynucleotides that are suitable for carrying out a specific amplificationreaction. Thus the primers used according to the present invention maybe nucleic acid oligomers of at least 15 consecutive nucleotides with asequence identity of at least 80% compared to the human transketolaselike-1 gene sequence or may be complementary or reverse complementary tosuch a sequence. The primers according to the present inventionspecifically hybridise to the sequence disclosed herein or a partthereof under conditions suitably applied in the course of a nucleicacid amplification reaction but do not hybridise to an othertransketolase or transketolase like sequence. Antisense oligonucleotidesas used herein may be nucleic acid molecules reverse complementary tothe transcripts of the disclosed coding sequence, that are able to bindto the transcripts by base pairing and such inhibit or reduce expressionof said coding sequence.

The nucleic acids used according to the present invention may also bechemically pre-treated nucleic aids. Such chemically pre-treated nucleicacids may comprise any nucleic acid as disclosed herein, which has beentreated with a chemical agent suitable to result in modifications in thenucleic acid molecules. Said modifications may for example comprisespecific modifications of particular bases within the nucleic acid. Suchchemical treatments may comprise treatment with e.g. sodium bisulphite,hydrazine or potassium permanganate. The sequences of special interestin experiments using chemical pre-treatment of nucleic acids may forexample comprise coding or non-coding regions of the sequences. Examplesof non-coding regions that may be treated by chemicals are promoterregions or CpG islands in 5′ UTRs.

Human transketolase like-1 polypeptides as used according to the presentinvention may comprise amino acid chains of any length, including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds. Thus, a polypeptide comprising a portion of one of theabove human transketolase like-1 proteins, e.g. a protein comprising theamino acid sequence of human transketolase like-1 protein, may consistentirely of the portion, or the portion may be present within a largerpolypeptide that contains additional sequences. The additional sequencesmay be derived from the native protein or may be heterologous, and suchsequences may (but need not) be immunoreactive and/or antigenic. Asdetailed below, such polypeptides may be isolated from tumor tissue orprepared by synthetic or recombinant means.

As used herein, a polypeptide exhibiting biological properties of thehuman transketolase like-1 polypeptide is understood to be a polypeptidehaving at least one of the activities, such as enzymatic activities(transketolase activity), inter protein interaction activities,responsiveness of the enzymatic activity to thiamine presence, orantigenic or immunogenic properties e.g. capability of binding anantibody directed against said polypeptide (i.e. comprising animmunogenic portion) of said human transketolase like-1 polypeptide.

Immunogenic portion as used herein is a portion of a protein that isrecognized by a B-cell and/or T-cell surface antigen receptor. Theimmunogenic portions comprise at least 5 amino acid residues, morepreferably at least 10 amino acid residues and most preferably at least15 amino acid residues of the protein disclosed herein. In a preferredembodiment of the present invention, particular domains of the protein,such as for example transmembrane domains or N-terminal leader sequenceshave been deleted.

The immunogenic portions according to the present invention react withantisera or specific antibodies in the same or nearly same intensity asthe native full length proteins. The immunogenic portions are generallyidentified using the techniques well known in the art. Possibletechniques are for example screening of the polypeptides for the abilityto react with antigen-specific antibodies, antisera and/or T-cell linesor clones.

The human transketolase like-1 polypeptides used according to thepresent invention comprise also variants of the native proteins. Thesevariants may differ from the native protein in one or more alterationssuch as substitutions, deletions, additions and/or insertions. Theimmunoreactivity and or biological activity of the variants according tothe present invention is not substantially diminished compared to thenative proteins. In a preferred embodiment of the invention theimmunoreactivity and or activity is diminished less than 50%, in a morepreferred embodiment the immunoreactivity and or activity is diminishedless than 20% compared to the native polypeptides. In another preferredembodiment of the present invention the variants of the polypeptides maybe varied, such that the activity of the native protein is increased,decreased or lost. These variants may for example be employed in thetherapy of disorders associated with the overexpression of humantransketolase like-1 gene. In a preferred embodiment variants may bedeficient in one or more portions, such as for example N-terminal leadersequences, transmembrane domains or small N- and/or C-terminalsequences. The variants exhibit preferably 70%, more preferably at least90% and most preferably at least 95% identity to the polypeptidesdisclosed according to the present invention.

The variants used according to the present invention comprise preferablyconservative substitutions, so that the amino acids changed aresubstituted for amino acids with similar properties. The propertiesconcerned may include polarity, charge, solubility, hydrophobicity,hydrophilicity and/or amphipathic nature of the amino acid residues.

The variants used according to the invention may also compriseadditional terminal leader sequences, linkers or sequences, which enablesynthesis, purification or stability of the polypeptides in an easier ormore comfortable way.

Variants of the polypeptides used in the methods according to thepresent invention may be produced by means of conventional molecularbiological processes (see, e.g., Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2nd edition Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.) For example it is possible to introducedifferent mutations into the nucleic acid molecules of the invention. Asa result a human transketolase like-1 polypeptide with possibly modifiedbiological properties may be synthesized. One possibility is theproduction of deletion mutants in which nucleic acid molecules areproduced by continuous deletions from the 5′- or 3′-terminal of thecoding DNA sequence and that lead to the synthesis of polypeptides thatare shortened accordingly. Another possibility is the introduction ofsingle-point mutations at positions where a modification of the aminoacid sequence influences, e.g., the enzyme activity or the regulation ofthe enzyme. By this method muteins can be produced, for example, thatpossess a modified Km-value or that are no longer subject to theregulation mechanisms that normally exist in the cell, e.g. with regardto allosteric regulation or covalent modification. Such muteins mighte.g. be valuable as therapeutically useful compounds, e.g. antagonists.

For the manipulation in prokaryotic cells by means of geneticengineering the nucleic acid molecules used for the methods of theinvention or parts of these molecules can be introduced into plasmidsallowing a mutagenesis or a modification of a sequence by recombinationof DNA sequences. By means of conventional methods (cf. Sambrook et al.,supra) bases may be exchanged and natural or synthetic sequences may beadded. In order to link the DNA fragments with each other, adapters orlinkers may be added to the fragments. Furthermore, manipulations may beperformed that provide suitable cleavage sites or that removesuperfluous DNA or cleavage sites. If insertions, deletions orsubstitutions are possible, in vitro mutagenesis, primer repair,restriction or ligation may be performed. As analysis method usuallysequence analysis, restriction analysis and other biochemical ormolecular biological methods may be used.

The polypeptides may comprise fusion or chimeric polypeptides containingsequences disclosed herein. Fusion proteins comprise the inventivepolypeptide, a portion thereof or variants of the inventive polypeptideor portions thereof together with any second and further polypeptides,such as once more the inventive polypeptide, a portion thereof orvariants of the inventive polypeptide or portions thereof and/or anyheterologous polypeptides. Heterologous polypeptides may compriseenzymes, receptor molecules, antigens, antigenic or immunogenic epitopesor fragments thereof, antibodies or fragments thereof, signallingpolypeptides or signal transducing polypeptides etc. The immunogenicprotein may for example be capable of eliciting a recall response.Examples of such proteins include tetanus, tuberculosis and hepatitisproteins (see, for example, Stoute et al. New Engl. J. Med., 336:86-91(1997)). In one embodiment of the invention the fusion peptides may beconstructed for enhanced detection or purification of the polypeptides.For the purpose of purification tags, such as e.g. his-tags, myc-tagsetc. may be added to the polypeptides. For the purpose of detectionantigenic portions, enzymes, chromogenic sequences etc. may be fused tothe polypeptides. The fusion proteins of the present invention may (butneed not) include a linker peptide between the first and secondpolypeptides.

A nucleic acid sequence encoding a fusion protein used in the presentinvention is constructed using known recombinant DNA techniques toassemble separate nucleic acid sequences encoding the first and secondpolypeptides into an appropriate expression vector. The 3′ end of anucleic acid sequence encoding the first polypeptide is ligated, with orwithout a peptide linker, to the 5′ end of a nucleic acid sequenceencoding the second polypeptide ensuring the appropriate reading framesof the sequences to permit mRNA translation of the two nucleic acidsequences into a single fusion protein that retains the biologicalactivity of both the first and the second polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptides by a distance sufficient to ensure, that eachpolypeptide folds into its secondary and tertiary structures. Such apeptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may be from 1 to about 50 amino acids in length.Peptide sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The human transketolase like-1 polypeptides for use in the methodaccording to the present invention and nucleic acids encoding suchpolypeptides, may be isolated from tumor tissue using any of a varietyof methods well known in the art. Nucleic acid sequences correspondingto a gene (or a portion thereof) encoding one of the inventive tumorpolypeptides may be isolated from a tumor cDNA library using asubtraction technique. Partial nucleic acid sequences thus obtained maybe used to design oligonucleotide primers for the amplification offull-length nucleic acid sequences from a human genomic nucleic acidlibrary or from a tumor cDNA library in a polymerase chain reaction(PCR), using techniques well known in the art (see, for example, Mulliset al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; Erlich ed.,PCR Technology, Stockton Press, New York, 1989). For this approach,sequence-specific primers may be designed based on the nucleotidesequences provided herein and may be purchased or synthesized.

The human transketolase like-1 polypeptides used for the methoddisclosed herein may also be generated by synthetic means. Inparticular, synthetic polypeptides having fewer than about 100 aminoacids, and generally fewer than about 50 amino acids, may be generatedusing techniques well known to those of ordinary skill in the art. Forexample, such polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain (see, for example, Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963). Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

The ligated nucleic acid sequences encoding the polypeptides used forthe methods disclosed herein are operably linked to suitabletranscriptional or translational regulatory elements known to the personskilled in the art. The regulatory elements responsible for expressionof nucleic acid may be located e.g. 5′ to the nucleic acid sequenceencoding the first polypeptides, within the coding sequences or 3′ tothe nucleic acid sequences encoding the first or any furtherpolypeptide. Stop codons required to end translation and transcriptiontermination signals are present 3′ to the nucleic acid sequence encodingthe second polypeptide.

The polypeptides used for the methods according to the present inventionmay be isolated. This means that the molecules may be removed from theiroriginal environment. Naturally occurring proteins are isolated if theyare separated from some or all of the materials, which coexist in thenatural environment. Polynucleotides are isolated for example if theyare cloned into vectors.

In certain preferred embodiments, described in more detail below, thepolypeptides used in a method as disclosed herein may be prepared in anisolated, substantially pure form (i.e., the polypeptides are homogenousas determined by amino acid composition and primary sequence analysis).Preferably, the polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. The substantially pure polypeptides may for example beemployed in pharmaceutical compositions.

Furthermore the present invention makes use of binding agents thatspecifically bind to a human transketolase like-1 protein. These bindingagents may comprise for example antibodies and antigen-bindingfragments, bifunctional hybrid antibodies, peptidomimetics containingminimal antigen-binding epitopes etc.

An antibody or antigen-binding agent is said to react specifically, ifit reacts at a detectable level with a protein used herein, and does notsignificantly react with other proteins. The antibodies according to thepresent invention may be monoclonal or polyclonal antibodies. As usedherein, the term antibody or monoclonal antibody is meant to includeintact molecules as well as antibody fragments (such as, for example,Fab and F(ab′)2 fragments), which are capable of specifically binding toprotein. Fab and F(ab′)2 fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., J.Nucl. Med. 24:316-325 (1983). Thus, these fragments are preferred, aswell as the products of a Fab or other immunoglobulin expressionlibrary. Moreover, antibodies used in the present invention includechimeric, single chain, and humanized antibodies.

According to the present invention binding agents may be used isolatedor in combination. By means of combination it is possible to achieve ahigher degree of sensitivity. The term antibody, preferably, relates toantibodies, which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations.

Monoclonal antibodies are made from an antigen containing fragments ofthe polypeptide used in the invention using any of a variety oftechniques known to those of ordinary skill in the art; see, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In one such technique, an immunogen comprising theantigenic polypeptide or a synthetic part thereof is initially injectedinto any of a wide variety of mammals (e.g., mice, rats, rabbits, sheepand goats). In this step, the polypeptides of this invention may serveas the immunogen without modification. Alternatively, particularly forrelatively short polypeptides, a superior immune response may beelicited if the polypeptide is joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. The immunogen isinjected into the animal host, preferably according to a predeterminedschedule incorporating one or more booster immunizations, and theanimals are bled periodically. Polyclonal antibodies specific for thepolypeptide may then be purified from such antisera by, for example,affinity chromatography using the polypeptide coupled to a suitablesolid support.

Monoclonal antibodies specific for the antigenic polypeptide of interestmay be prepared, for example, using the technique of Köhler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a non-ionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The human transketolase like-1polypeptides may be used in the purification process in, for example, anaffinity chromatography step.

The human transketolase like-1 specific antibodies used according to thepresent invention may comprise further binding sites for eithertherapeutic agents or other polypeptides or may be coupled to saidtherapeutic agents or polypeptides. Therapeutic agents may comprisedrugs, toxins, radio nuclides and derivatives thereof. The agents may becoupled to the binding agents either directly or indirectly for exampleby a linker or carrier group. The linker group may for example functionin order to enable the coupling reaction between binding agent andtherapeutic or other agent or the linker may act as a spacer between thedistinct parts of the fusion molecule. The linker may also be cleavableunder certain circumstances, so as to release the bound agent under saidconditions. The therapeutic agents may be covalently coupled to carriergroups directly or via a linker group. The agent may also benon-covalently coupled to the carrier. Carriers that can be usedaccording to the present invention are for example albumins,polypeptides, polysaccharides or liposomes.

The human transketolase like-1 specific antibodies used according to thepresent invention may be coupled to one or more agents. The multipleagents coupled to one antibody may be all of the same species or may beseveral different agents bound to one antibody.

The methods disclosed herein are applicable to all eukaryotic organismsprone to be affected by disorders associated with the overexpression oftransketolase like-1 gene. Individuals as used in the context of thepresent invention may for example comprise mammals, such as animals ofagricultural interest (cows, sheep, horses, pigs, etc.), companionanimals (cats, dogs, etc.), animals commonly employed for research use(rats, mice, hamsters, etc.) or human beings.

Diagnosis as used in the context of the present invention may comprisedetermining the level of human transketolase like-1 gene products in asample. Based upon the determined level of human transketolase like-1gene products in the samples individuals can be subdivided intosubgroups. The subgroups may be created according to clinical data, suchas e.g. survival, recurrence of disease, frequency of metastases etc.,related to the particular level of transketolase like-1 gene productsdetermined in the samples.

Based upon these subgroups an assessment of prognosis may be done.According to the subgroups the therapy of the individuals affected bythe tumors may be tailored.

For example the overexpression of transketolase like-1 gene and anenhanced activity of the pentose-phosphate cycle in a subset of colon,stomach, pancreas and lung tumors suggest a mechanism by which thiamine(vitamin B1) promotes nucleic acid ribose synthesis and tumor cellproliferation via the nonoxidative transketolase pathway. Therefore thethiamine intake of cancer patients has direct consequences for thegrowth rate of tumors with an overexpression of the transketolase like-1gene. This provides also background information and helps to developguidelines for alternative treatments with antithiamine transketolaseinhibitors in the clinical setting. Clinical and experimental datademonstrate increased thiamine utilization of human tumors and itsinterference with experimental chemotherapy. Analysis of RNA riboseindicates that glucose carbons contribute to over 90% of ribosesynthesis in cultured cervix and pancreatic carcinoma cells and thatribose is synthesized primarily through the thiamine dependenttransketolase pathway (>70%). Antithiamine compounds significantlyinhibit nucleic acid synthesis and tumor cell proliferation in vitro andin vivo in several tumor models. The medical literature reveals littleinformation regarding the role of the thiamine dependent transketolasereaction in tumor cell ribose production, which is a central process inde novo nucleic acid synthesis and the salvage pathways for purines.

As thiamine dependent transketolase pathway is the central avenuesupplying ribose phosphate for nucleic acids in tumors an excessivethiamine supplementation may be responsible for failed therapeuticattempts to terminate cancer cell proliferation. The detection of asubset of colon and lung tumors with an overexpression of thetransketolase like-1 gene provides an important step to anindividualized cancer therapy and limited administration of thiamine andconcomitant treatment with transketolase inhibitors is a more rationalapproach to treat cancer.

Thus based on the detection of overexpression of TKT-L1 new treatmentstrategies may be tailored targeting specific biochemical reactions ofpentose-phosphate cycle by hormones related to glucose metabolism,controlling thiamine intake, the cofactor of the nonoxidativetransketolase pentose-phosphate cycle reaction, or treating cancerpatients with antithiamine analogues.

Thus in one embodiment of the invention disorders characterized byoverexpression of human transketolase like-1 gene may be treated inaccordance to the level of overexpression of transketolase like-1 gene.Using the non-oxidative pentose-phosphate cycle reactions to inhibitglucose utilizing pathways selectively for nucleic acid productionoffers a new target site for cancer treatment with a strong regulatoryeffect on the cell cycle.

In one embodiment the treatment of disorders associated withoverexpression of transketolase like-1 gene may comprise restrictedadministration of thiamine to the affected individuals. In anotherembodiment the treatment may comprise the administration oftransketolase inhibitors, such as e.g. antithiamine compounds.

Monitoring may comprise detecting the level of human transketolaselike-1 gene products in samples taken at different points in time anddetermining the changes in said level. According to said changes thecourse of the disease can be followed. The course of the disease may beused to select therapy strategies for the particular individual.

Another aspect of diagnosis and monitoring of the disease courseaccording to the present invention may comprise the detection of minimalresidual disease. This may comprise for example the detection of a humantransketolase like-1 gene products level in one or more body samplesfollowing initial therapy of an individual once or at several timepoints. According to the level of human transketolase like-1 geneproducts detected in the samples one may select a suitable therapy forthe particular individual.

In another preferred embodiment the diagnostic method is carried out todetect disseminated tumor cells in biological samples as diagnosis ofminimal residual disease (MRD).

Disorders characterized by abnormal cell proliferation, as used in thecontext of the present invention, may comprise for example neoplasmssuch as benign and malignant tumors, carcinomas, sarcomas, leukemias,lymhomas or dysplasias. Tumors may comprise tumors of the head and theneck, tumors of the respiratory tract, tumors of the gastrointestinaltract, tumors of the urinary system, tumors of the reproductive system,tumors of the endocrine system, tumors of the central and peripheralnervous system, tumors of the skin and its appendages, tumors of thesoft tissues and bones, tumors of the lymphopoietic and hematopoieticsystem etc.

In a preferred embodiment the tumor is for example cancer of the of thehead and the neck, cancer of the respiratory tract, cancer of thegastrointestinal tract, cancer of the skin and its appendages, cancer ofthe central and peripheral nervous system, cancer of the urinary system,cancer of the reproductive system, cancer of the endocrine system,cancer of the soft tissues and bone, cancer of the hematopoietic andlymphopoietic system. In the most preferred embodiment of the inventionthe carcinoma is cervical cancer, colon cancer, gastric cancer, breastcancer, bladder cancer etc.

The tumors according to the present invention may comprise tumors, whichshow detectable lymph-node involvement (node positive tumors) as well astumors, without detectable spread to lymph-nodes (node negative tumors).In one embodiment of the invention the gastrointestinal tumors aretumors without detectable spread to lymph nodes.

A sample according to the method of the present invention may compriseany sample comprising cells or cell debris. Samples may comprise samplesof clinical relevance, such as e.g. secretions, such as gastric juice,bile or pancreatic juice, smears, body fluids, such as serum, blood,plasma urine, semen, stool, biopsies or cell- and tissue-samples.Biopsies as used in the context of the present invention may comprisee.g. resection samples of tumors, tissue samples prepared by endoscopicmeans or needle biopsies of organs. Furthermore any sample potentiallycontaining the marker molecules to be detected may be a sample accordingto the present invention.

Such samples may comprise for example intact cells, lysed cells or anyliquids containing proteins, peptides or nucleic acids. Even solids, towhich cells, cell fragments or marker molecules, such as humantransketolase like-1 nucleic acids or human transketolase like-1proteins, may adhere may be samples according to the present invention.Such solids may comprise for example membranes, glass slides, beads etc.

Preparation of a sample may comprise e.g. obtaining a sample of atissue, a body fluid, of cells, of cell debris from a patient. Accordingto the present invention preparation of the sample may also compriseseveral steps of further preparations of the sample, such as preparationof dissections, preparation of lysed cells, preparation of tissuearrays, isolation of polypeptides or nucleic acids, preparation of solidphase fixed peptides or nucleic acids or preparation of beads, membranesor slides to which the molecules to be determined are coupled covalentlyor non-covalently.

The method for detection of the level of the human transketolase like-1gene product according to the present invention is any method, which issuited to detect very small amounts of specific biologically activemolecules in biological samples. The detection reaction according to thepresent invention is a detection either on the level of nucleic acids oron the level of polypeptides.

The detection may be carried out in solution or using reagents fixed toa solid phase. The detection of one or more molecular markers, such aspolypeptides or nucleic acids, may be performed in a single reactionmixture or in two or separate reaction mixture. Alternatively thedetention reactions for several marker molecules may for example beperformed simultaneously in multi-well reaction vessels. The markerscharacteristic for the human transketolase like-1 gene products may bedetected using reagents that specifically recognise these molecules. Thedetection reaction for the marker molecules may comprise one or morereactions with detecting agents either recognizing the initial markermolecules or recognizing the prior molecules used to recognize othermolecules.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the humantransketolase like-1 gene markers. The reporter reaction may be forexample a reaction producing a coloured compound, a bioluminescencereaction, a fluorescence reaction, generally a radiation emittingreaction etc. In a preferred embodiment, different marker molecules maybe recognized by agents that produce different reporter signals, so thatthe signals referring to marker molecules could be distinguished.

Applicable formats for the detection reaction according to the presentinvention may be, blotting techniques, such as Western-Blot,Southern-blot, Northern-blot. The blotting techniques are known to thoseof ordinary skill in the art and may be performed for example aselectro-blots, semidry-blots, vacuum-blots or dot-blots. Amplificationreaction may also be applicable for the detection of e.g. nucleic acidmolecules. Furthermore immunological methods for detection of moleculesmay be applied, such as for example immunoprecipitation or immunologicalassays, such as ELISA, RIA, lateral flow assays, immuno-cytochemicalmethods etc.

In one preferred embodiment of the invention the detection of the levelof human transketolase like-1 gene products is carried out by detectionof the level of nucleic acids coding for the human transketolase like-1gene products or fragments thereof present in the sample. The means fordetection of nucleic acid molecules are known to those skilled in theart. The procedure for the detection of nucleic acids can for example becarried out by a binding reaction of the molecule to be detected tocomplementary nucleic acid probes, proteins with binding specificity forthe nucleic acids or any other entities specifically recognizing andbinding to said nucleic acids. This method can be performed as well invitro as directly in situ for example in the course of a detectingstaining reaction. Another way of detecting the human transketolaselike-1 gene products in a sample on the level of nucleic acids performedin the method according to the present invention is an amplificationreaction of nucleic acids, which can be carried out in a quantitativemanner such as for example the polymerase chain reaction. In a preferredembodiment of the present invention real time RT PCR may be used toquantify the level of transketolase like-1 RNA in samples of tumors.

In another preferred embodiment of the invention the detection of thelevel of human transketolase like-1 gene products is carried out bydetermining the level of expression of a protein. The determination ofthe human transketolase like-1 gene products on the protein level canfor example be carried out in a reaction comprising an antibody specificfor the detection of the human transketolase like-1 protein. Theantibodies can be used in many different detection techniques forexample in Western-blot, ELISA or immunoprecipitation. Generallyantibody based detection can be carried out as well in vitro as directlyin situ for example in the course of an immuno-histochemical stainingreaction. Any other method for determining the amount of particularpolypeptides in biological samples can be used according to the presentinvention.

In one preferred embodiment of the invention the level of humantransketolase like-1 gene products is significantly elevated compared toa control test sample. In this case the human transketolase like-1 geneis overexpressed in the sample.

One example for the diagnosis of disorders associated with theexpression of the human transketolase like-1 gene may comprise thedetection of auto-antibodies directed against polypeptides encoded bythe human transketolase like-1 gene. The polypeptides used for themethods according to the present invention may be used to detect thepresence or absence of such antibodies in body fluids by methods knownto those of skill in the art.

In one preferred embodiment the detection of tissues expressingtransketolase like-1 gene products is carried out in form of molecularimaging procedures. The respective procedures are known to those ofordinary skill in the art. Imaging methods for use in the context of thepresent invention may for example comprise MRI, SPECT, PET and othermethods suitable for in vivo imaging.

In one embodiment the method may be based on the enzymatic conversion ofinert or labelled compounds to molecules detectable in the course ofmolecular imaging methods by the transketolase like-1 molecules. Inanother embodiment the molecular imaging method may be based on the useof compounds carrying a suitable label for in vivo molecular imaging,such as radio isotopes, metal ions etc., specifically binding totransketolase like-1 molecules in vivo.

In a preferred embodiment of the invention these compounds are non-toxiccompounds and may be eliminated from the circulation of organisms, suchas humans, in a time span, that allows for performing the detection oflabel accumulated in tumor tissue overexpressing transketolase like-1gene. In another preferred embodiment of the invention compounds areused for molecular imaging, for which clearance from the circulation isnot relevant for performing the molecular imaging reaction. This may befor example due to low background produced by the circulating moleculesetc. The compounds for use in molecular imaging methods are administeredin pharmaceutical acceptable form in compositions that may additionallycomprise any other suitable substances, such as e.g. otherdiagnostically useful substances, therapeutically useful substances,carrier substances or the like.

Another aspect of the present invention is a testing kit for performingthe method according to the present invention. The kit may be forexample a diagnostic kit or a research kit.

A kit according to the present invention comprises at least an agentsuitable for detecting the molecules disclosed herein. Furthermore a kitaccording to present invention may comprise:

a) reagents for the detection of the human transketolase like-1 geneproductsb) reagents and buffers commonly used for carrying out the detectionreaction, such as buffers, detection-markers, carrier substances andothersc) a human transketolase like-1 sample for carrying out a positivecontrol reaction

The reagent for the detection of the human transketolase like-1 gene mayinclude any agent capable of binding to the human transketolase like-1molecule. Such reagents may include proteins, polypeptides, nucleicacids, peptide nucleic acids, glycoproteins, proteoglycans,polysaccharides or lipids.

The human transketolase like-1 sample for carrying out a positivecontrol may comprise for example human transketolase like-1 nucleicacids or polypeptides or fragments thereof in applicable form, such assolution or salt, peptides in applicable form, tissue section samples orpositive cells.

In a preferred embodiment of the invention the detection of the humantransketolase like-1 gene product is carried out on the level ofpolypeptides. In this embodiment the binding agent may be for example anantibody specific for the human transketolase like-1 or a fragmentthereof.

In an other embodiment of the test kit the detection of the humantransketolase like-1 gene products is carried out on the nucleic acidlevel. In this embodiment of the invention the reagent for the detectionmay be for example a nucleic acid probe or a primerreverse-complementary to said human transketolase like-1 nucleic acid.

In a further aspect the present invention relates to the use of one ormore of the compounds useful for the methods according to the presentinvention such as a nucleic acid molecule, a recombinant vector, apolypeptide, an antisense RNA sequence, a ribozyme or an antibody forthe preparation of a pharmaceutical composition for the treatment ofcancer, preferably colon cancer, pancreatic carcinoma, gastric cancer,lung cancer.

The polypeptides, polynucleotides and binding agents useful in themethod according to the present invention may be incorporated intopharmaceutical or immunogenic compositions. The pharmaceuticalcompositions comprise said compounds and a physiologically acceptablecarrier.

A pharmaceutical composition or vaccine may for example contain DNA thatcodes for one or more polypeptides according to the present invention.The DNA may be administered in a way that allows the polypeptides to begenerated in situ. Suitable expression systems are known to thoseskilled in the art. The expression of the polypeptides may for examplebe persistent or transient. In pharmaceutical compositions and/orvaccines, providing for in-situ expression of polypeptides, the nucleicacids may be present within any suitable delivery system known to thoseof ordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Appropriate nucleic acidexpression systems comprise the necessary regulatory nucleic acidsequences for expression in the patient, such as suitable promoters,terminators etc. Bacterial delivery systems may for example employ theadministration of a bacterium that expresses an epitope of a cellantigen on its cell surface. In a preferred embodiment, the nucleic acidmay be introduced using a viral expression system such as e.g., vacciniavirus, retrovirus, or adenovirus, which may involve the use of anon-pathogenic, replication competent virus. Suitable systems aredisclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321, 1989;Flexner et al., Ann. N.Y. Acad Sci. 569:86-103, 1989; Flexner et al.,Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988;Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzmanet al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.73:1202-1207, 1993. In another embodiment transgenic mammalian cells maybe used for delivery and/or expression of the nucleic acids. The methodsfor producing nucleic acid constructs suitable for in-situ expression ofpolypeptides are known to those of skill in the art.

In another embodiment of the invention the nucleic acids may be forexample anti-sense constructs.

The nucleic acid may also be administered as a naked nucleic acid. Inthis case appropriate physical delivery systems, which enhance theuptake of nucleic acid may be employed, such as coating the nucleic acidonto biodegradable beads, which are efficiently transported into thecells. Administration of naked nucleic acids may for example be usefulfor the purpose of transient expression within a host or host cell.

Alternatively the pharmaceutical compositions may comprise one or morepolypeptides. The polypeptides incorporated into pharmaceuticalcompositions may be the human transketolase like-1 polypeptides incombination with one or more other known polypeptides such as forexample enzymes, antibodies, regulatory factors, such as cyclins,cyclin-dependent kinases or CKIs, or toxins.

The pharmaceutical compositions may be administered by any suitable wayknown to those of skill in the art. The administration may for examplecomprise injection, such as e.g., intracutaneous, intramuscular,intravenous or subcutaneous injection, intranasal administration forexample by aspiration or oral administration. A suitable dosage toensure the pharmaceutical benefit of the treatment should be chosenaccording the parameters, such as age, sex, body weight etc. of thepatient, known to those of skill in the art.

The type of carrier to be employed in the pharmaceutical compositions ofthis invention, will vary depending on the mode of administration. Forparenteral administration, such as subcutaneous injection, the carrierpreferably comprises water, saline, alcohol, a lipid, a wax and/or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and/or magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticglycolide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

The compounds of the present invention may furthermore be incorporatedinto immunogenic compositions.

The constituents of an immunogenic composition may comprise vaccines,antigens, antigenic fragments or nucleic acids coding for antigens orantigenic fragments to be expressed in situ. This compounds may bepresent polypeptides, or as nucleic acids, that allow the polypeptidesto be expressed in situ. Immunogenic compositions comprise saidcompounds and additionally an immunostimulant or immunogenic adjuvant.

Polypeptides of the present invention or fragments thereof, thatcomprise an immunogenic portion of a human transketolase like-1 protein,may be used in immunogenic compositions, wherein the polypeptide e.g.stimulates the patient's own immune response to tumor cells. A patientmay be afflicted with disease, or may be free of detectable disease.Accordingly, the compounds disclosed herein may be used to treat canceror to inhibit the development of cancer. The compounds may beadministered either prior to or following a conventional treatment oftumors such as surgical removal of primary tumors, treatment byadministration of radiotherapy, conventional chemotherapeutic methods orany other mode of treatment of the respective cancer or its precursors.Immunogenic compositions such as vaccines may comprise one or morepolypeptides and a non-specific immune-response enhancer, wherein thenon-specific immune response enhancer is capable of eliciting orenhancing an immune response to an exogenous antigen. Examples ofnon-specific immune response enhancers include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and for example liposomes intowhich the polypeptide may be incorporated. Pharmaceutical compositionsand vaccines may also contain other epitopes of tumor antigens, eitherincorporated into a fusion protein as described above (i.e., a singlepolypeptide that contains multiple epitopes) or present within aseparate polypeptide.

Any suitable immune-response enhancer may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminium hydroxide or mineral oil, and anon-specific stimulator of immune response, such as lipid A, Bordetellapertussis or Mycobacterium tuberculosis. Such adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65(Merck and Company, Inc., Rahway, N.J.).

For therapeutic purposes polypeptides, polynucleotides or binding agentsmay be administered in a variety of ways. Possible ways may for examplecomprise intracutaneous, intramuscular, intravenous or subcutaneousinjection, intranasal administration for example by aspiration or oraladministration.

Another aspect of the present invention is to provide a method fortherapy and/or vaccination. According to the present invention a therapyof cell proliferative disorders can be carried out using humantransketolase like-1 polypeptides and/or polynucleotides. The therapymay be for example immunotherapy or somatic gene therapy.

The human transketolase like-1 polypeptides and/or polynucleotides mayaccording to the present invention be used for vaccination against cellproliferative disorders. Vaccination according to the present inventionmay comprise administering an immunogenic compound to an individual forthe purpose of stimulating an immune response directed against saidimmunogenic compound and thus immunizing said individual against saidimmunogenic compound. Stimulating an immune response may compriseinducing the production of antibodies against said compound as well asstimulating cytotoxic T-cells. For the purpose of vaccination thepolypeptides, nucleic acids and binding agents according to the presentinvention may be administered in a physiological acceptable form. Thecomposition to be administered to individuals may comprise one or moreantigenic components, physiologically acceptable carrier substances orbuffer solutions, immunostimulants and/or adjuvants. Adjuvants maycomprise for example Freund's incomplete adjuvant or Freund's completeadjuvant or other adjuvants known to those of skill in the art.

The composition may be administered in any applicable way such as e.g.intravenous, subcutaneous, intramuscular etc. The dosage of thecomposition depends on the particular case and purpose of thevaccination. It has to be adapted to parameters by the individualtreated such as age, weight, sex etc. Furthermore the type of the immuneresponse to be elicited has to be taken into account. In general it maybe preferable if an individual receives 100 μg-1 g of a polypeptideaccording to the present invention or 10⁶-10¹² MOI of a recombinantnucleic acid, containing a nucleic acid according to the presentinvention in a form that may be expressed in situ.

Individuals for the purpose of vaccination may be any organismscontaining transketolase like-1 proteins and being able get affected bycell proliferative disorders.

Vaccination of individuals may be favourable e.g. in the case ofaltered, non wild-type sequences or structure of marker moleculesassociated with cell proliferative disorders.

Polypeptides disclosed herein may also be employed in adoptiveimmunotherapy for the treatment of cancer. Adoptive immunotherapy may bebroadly classified into either active or passive immunotherapy. Inactive immunotherapy, treatment relies on the in vivo stimulation of theendogenous host immune system to react against tumors with theadministration of immune response-modifying agents (for example, tumorvaccines, bacterial adjuvants, and/or cytokines).

In passive immunotherapy, treatment involves the delivery of biologicreagents with established tumor-immune reactivity (such as effectorcells or antibodies) that can directly or indirectly mediate antitumoreffects and does not necessarily depend on an intact host immune system.Examples of effector cells include T lymphocytes (for example, CD8+cytotoxic T-lymphocyte, CD4+ T-helper, tumor-infiltrating lymphocytes),killer cells (such as Natural Killer cells, lymphokine-activated killercells), B cells, or antigen presenting cells (such as dendritic cellsand macrophages) expressing the disclosed antigens. The polypeptidesdisclosed herein may also be used to generate antibodies oranti-idiotypic antibodies (as in U.S. Pat. No. 4,918,164), for passiveimmunotherapy.

The predominant method of procuring adequate numbers of T-cells foradoptive immunotherapy is to grow immune T-cells in vitro. Cultureconditions for expanding single antigen-specific T-cells to severalbillion in number with retention of antigen recognition in vivo are wellknown in the art. These in vitro culture conditions typically utilizeintermittent stimulation with antigen, often in the presence ofcytokines, such as IL-2, and non-dividing feeder cells. As noted above,the immunoreactive polypeptides described herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate sufficientnumber of cells for immunotherapy. In particular, antigen-presentingcells, such as dendritic, macrophage or B-cells, may be pulsed withimmunoreactive polypeptides or transfected with a nucleic acidsequence(s), using standard techniques well known in the art. Forexample, antigen presenting cells may be transfected with a nucleic acidsequence, wherein said sequence contains a promoter region appropriatefor increasing expression, and can be expressed as part of a recombinantvirus or other expression system. For cultured T-cells to be effectivein therapy, the cultured T-cells must be able to grow and distributewidely and to survive long term in vivo. Studies have demonstrated thatcultured T-cells can be induced to grow in vivo and to survive long termin substantial numbers by repeated stimulation with antigen supplementedwith IL-2 (see, for example, Cheever, M., et al, “Therapy With CulturedT Cells: Principles Revisited,” Immunological Reviews, 157:177, 1997).

The polypeptides disclosed herein may also be employed to generateand/or isolate tumor-reactive T-cells, which can then be administered tothe patient. In one technique, antigen-specific T-cell lines may begenerated by in vivo immunization with short peptides corresponding toimmunogenic portions of the disclosed polypeptides. The resultingantigen specific CD8+ CTL clones may be isolated from the patient,expanded using standard tissue culture techniques, and returned to thepatient.

Alternatively, peptides corresponding to immunogenic portions of thepolypeptides of the invention may be employed to generate tumor reactiveT-cell subsets by selective in vitro stimulation and expansion ofautologous T-cells to provide antigen-specific T-cells which may besubsequently transferred to the patient as described, for example, byChang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells ofthe immune system, such as T-cells, may be isolated from the peripheralblood of a patient, using a commercially available cell separationsystem, such as CellPro Incorporated's (Bothell, Wash.) CEPRATE.™.system (see U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO89/06280; WO 91/16116 and WO 92/07243). The separated cells arestimulated with one or more of the immunoreactive polypeptides containedwithin a delivery vehicle, such as a microsphere, to provideantigen-specific T-cells. The population of tumor antigen-specificT-cells is then expanded using standard techniques and the cells areadministered back to the patient.

In another embodiment, T-cell and/or antibody receptors specific for thepolypeptides can be cloned, expanded, and transferred into other vectorsor effector cells for use in adoptive immunotherapy.

In a further embodiment, syngeneic or autologous dendritic cells may bepulsed with peptides corresponding to at least an immunogenic portion ofa polypeptide disclosed herein. The resulting antigen-specific dendriticcells may either be transferred into a patient, or employed to stimulateT-cells to provide antigen-specific T-cells, which may, in turn, beadministered to a patient. The use of peptide-pulsed dendritic cells togenerate antigen-specific T-cells and the subsequent use of suchantigen-specific T-cells to eradicate tumors in a murine model has beendemonstrated by Cheever et al, Immunological Reviews, 157:177, 1997.

Additionally, vectors expressing the disclosed nucleic acids may beintroduced into stem cells taken from the patient and clonallypropagated in vitro for autologous transplant back into the samepatient.

Monoclonal antibodies of the present invention may also be used astherapeutic compounds in order to diminish or eliminate tumors. Theantibodies may be used on their own (for instance, to inhibitmetastases) or coupled to one or more therapeutic agents. Suitableagents in this regard include radio nuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radio nuclides include90Y, 123I, 125I, 131I, 186Re, 188Re, 211At, and 212Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogs. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein.

Furthermore methods for treatment of disorders associated withoverexpression of transketolase like-1 gene may comprise any methodsuitable for the reduction of the activity of transketolase like 1polypeptide in an individual or in cells of an individual. These methodsmay comprise a reduction of the activity of transketolase like-1polypeptide by means of reduction of gene expression or by means ofreduction of enzymatic activity. Examples may comprise theadministration of antisense constructs, of ribozymes, of enzymeinhibitors, the administration of antagonists of cofactors oftransketolase like-1 polypeptides, as e.g. antithiamine compounds or thereduced administration of essential cofactors for the enzymatic activity(e.g. thiamine).

The methods for administration of ribozymes or antisense constructs areknown to those of skill in the art. The administration may take place asadministration of naked nucleic acids or as administration of nucleicacids that are suited for expression of the relevant active products insitu.

In one preferred embodiment the therapy of disorders associated with theoverexpression of transketolase like-1 gene comprises administration ofantithiamine compounds, or the reduction of thiamine uptake forindividuals showing disorders characterized by overexpression oftransketolase like-1 gene.

In a further embodiment, the present invention relates to a method ofidentifying and obtaining a drug candidate for therapy of colon,stomach, pancreatic or lung tumors comprising the steps of contacting aTKT-L1 polypeptide as used in the method of the present invention or acell expressing said polypeptide in the presence of components capableof providing a detectable signal in response to transketolase activity,to altered regulation of cell proliferation, and detecting presence orabsence of a signal or increase of the signal generated fromtransketolase activity or altered regulation of cell proliferation,wherein the absence or decrease of the signal is indicative for aputative drug.

The drug candidate may be a single compound or a plurality of compounds.The term “plurality of compounds” in a method of the invention is to beunderstood as a plurality of substances which may or may not beidentical.

Said compound or plurality of compounds may be chemically synthesized ormicrobiologically produced and/or comprised in, for example, samples,e.g., cell extracts from, e.g., plants, animals or microorganisms.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating TKT-L1 polypeptides.The reaction mixture may be a cell free extract or may comprise a cellor tissue culture. Suitable set ups for the method of the invention areknown to the person skilled in the art and are, for example, generallydescribed in Alberts et al., Molecular Biology of the Cell, thirdedition (1994) and in the appended examples. The plurality of compoundsmay be, e.g., added to the reaction mixture, culture medium, injectedinto cell or otherwise applied to the transgenic animal. The cell ortissue that may be employed in the method of the invention preferably isa host cell, mammalian cell or non-human transgenic animal of theinvention described in the embodiments hereinbefore.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating TKT-L1, or one canfurther subdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances of similar chemical and/orphysical properties, and most preferably said substances are identical.

The compounds which can be tested and identified according to a methodof the invention may be peptides, proteins, nucleic acids, antibodies,small organic compounds, hormones, peptidomimetics, PNAs or the like.

The compounds isolated by the above methods also serve as lead compoundsfor the development of analog compounds. The analogs should have astabilized electronic configuration and molecular conformation thatallows key functional groups to be presented to the TKT-L1 insubstantially the same way as the lead compound. In particular, theanalog compounds have spatial electronic properties which are comparableto the binding region, but can be smaller molecules than the leadcompound, frequently having a molecular weight below about 2 kD andpreferably below about 1 kD. Identification of analog compounds can beperformed through use of techniques such as self-consistent field (SCF)analysis, configuration interaction (CI) analysis, and normal modedynamics analysis. Computer programs for implementing these techniquesare available; e.g., Rein, Computer-Assisted Modeling of Receptor-LigandInteractions (Alan Liss, New York, 1989). Methods for the preparation ofchemical derivatives and analogues are well known to those skilled inthe art and are described in, for example, Beilstein, Handbook ofOrganic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, NewYork, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, N.Y., USA.Furthermore, said derivatives and analogues can be tested for theireffects according to methods known in the art; see also supra.Furthermore, peptidomimetics and/or computer aided design of appropriatederivatives and analogues can be used, for example, according to themethods described above.

Once the described compound has been identified and obtained, it ispreferably provided in a therapeutically acceptable form.

The present invention provides methods for detection and treatment ofdisorders characterized by abnormal cell proliferation, such as e.g.cancers. In one aspect the present invention provides a method for thedetection of disorders characterized by abnormal cell proliferation,such as e.g. cancers based on the determination of the presence orabsence and/or the level of expression of human transketolase like-1gene in biological samples. In a second aspect the present inventionprovides a method for treatment of disorders characterized by abnormalcell proliferation, such as e.g. cancers using human transketolaselike-1 gene products as therapeutically active agents. The inventionalso provides for therapeutic methods based on the reduction of theenzymatic activity of transketolase like-1 gene polypeptides. It is oneaspect of the invention to provide a method for rational tumormanagement based on the detection of transketolase like-1 gene productsin patient samples and the tailoring of a therapy correlated to thedetected overexpression of said gene products. Furthermore the presentinvention provides for a research or diagnostic test kit for performingthe reactions involved in the detection of the presence or absenceand/or the level of overexpression of human transketolase like-1 gene.Finally the present invention relates to pharmaceutical compositionsapplicable in the treatment of disorders according to the presentinvention.

The following examples are given for the purpose of illustration onlyand are not intended to limit the scope of the invention disclosedherein.

Example 1

Determining the Level of Human Transketolase like-1 mRNA Levels in ColonCarcinoma Tissues

Dissections of tumor biopsies can be semi-quantitatively analysed forthe mRNA level of human transketolase like-1 gene in an in-situ stainingreaction. The staining reaction is performed as follows:

The tissue dissections are incubated in ascending ethanol concentrationsup to 100% ethanol. After evaporation of the alcohol the dissections areboiled in 10 mM citrate buffer (pH 6.0) for pre-treatment of the tissue.The hybridisation mixture is prepared by mixing 50 μl of ready to usehybridisation buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 pmolof the probes. The probes are fluorescein-labelled oligonucleotides ofthe following sequence:

TCTCATCACAAGCAGCACAGGAC

The hybridisation mixture is heated to 95° C. and afterwardsequilibrated to 37° C. After the boiling procedure the dissections areincubated with each 50 μl of the hybridisation mixture for 2 hours at37° C. The dissections are washed in excess volumes of the wash bufferstwo times in 2×SSC at room temperature for 15 min and once in 1×SSC at50° C. for 15 min Then the dissections are rinsed two times at roomtemperature in 2×SSC. Following this washing procedure the dissectionsare incubated for 30 min with blocking buffer (NEN, Blockingpugger) atroom temperature. Then follows 1 hour incubation with a 1:100 diluted(in Blocking buffer, see above) Anti-Fluorescein-AP (DAKO A/S). Thedissections are then washed 2 times in 1×PBS/0,1% Tritonx100 for 10 minat room temperature, followed by one wash step with 1×PBS, 50 mM MgCl2(pH 9,2) for 10 min at room temperature.

Then the staining reaction is performed with NBT/BCIP (Sigma) for about30 min at room temperature. The staining reaction is stopped by a shortincubation with 1 mM EDTA in PBS. Finally the dissections are dipped inH₂O_(dest). and fixed with AquaTex (Merck). Then the stained dissectionscan be analysed microscopically.

The results show, that human transketolase like-1 gene is overexpressedin colon carcinoma tissue in comparison to normal colon tissue.

Example 2

Determination of Human Transketolase like-1 Gene and Transketolase Levelin Tissues of Carcinomas and Control Tissues using Semiquantitative RTPCR

Samples of colon carcinoma, adenocarcinoma of the lung and of carcinomasof the stomach are used to determine the level of human transketolaselike-1 mRNA and the level of human transketolase mRNA usingsemi-quantitative RT PCR. Tumor biopsies are used in this study.

Tumors are collected, snap frozen, and stored at −80° C. They areverified to be composed predominantly of neoplastic cells byhistopathological analysis. mRNA is isolated from tumors andpatient-matched normal tissue using Qiagen reagents (Qiagen, Hilden,Germany), and single-stranded cDNA is synthesized using Superscript II(Life Technologies, Inc.). Quantitative PCR is performed using the 7700Sequence Detector (Taqman™) and the SYBR Green PCR Master-Mix, asdescribed in the manufacturers manual (Applied Biosystems, Foster City,Calif.).

PCR reactions are performed in 25 μl volumes with a final concentrationof 300 nmol for each primer, with 95° C. for 15 sec and 60° C. for 60sec, for 40 cycles. The following primers are used for quantitative PCR:

Transketolase like-1: Primer A: CACCTTGGGATTCTGTGTGC Primer B:TCTCATCACAAGCAGCACAG Transketolase: Primer A: TGTGTCCAGTGCAGTAGTGGPrimer B: ACACTTCATACCCGCCCTAG

The specificity of the PCR products is verified by gel electrophoresis(data not shown).

The results show, that human transketolase like-1 gene is highlyoverexpressed in 1 out of 10 of colon carcinomas, in two out of five inlung adenocarcinomas and in three out of five carcinomas of the stomachin comparison to normal control tissue.

Especially the extent of overexpression of the transketolase like-1 genein the samples is noticeable. In total six out of 20 carcinomas showmore than eight fold overexpression of the TKT L-1 gene. In contrast thetransketolase gene in no case is significantly overexpressed.

The result shows, that in a subset of cancers of different originstransketolase like-1 gene is overexpressed. The transketolase gene incontrast is not differentially expressed in the tested tumor tissue.

Example 3

Immunochemical Detection of the Overexpression of tktl1 in TissueSamples of Carcinomas

Sections of formalin fixed, paraffin embedded gastric tissue sampleswere immunocytochemically stained using antibodies directed againsttktl1.

The sections were rehydrated through incubation in xylene and gradedethanol, and transferred to Aqua bidest. Antigen Retrieval was carriedout with 10 mM citrate buffer (pH 6.0) Therefore the slides were heatedin a waterbath for 40 min at 95° C. The slides were cooled down to RTfor 20 minutes, transferred to washing buffer (PBS/0.1% Tween20).

For inactivation of endogenous peroxidase the samples are incubated with3% H2O2 for 10 min at RT and afterwards washed in PBS/0.1% Tween20 for10 min.

The slides were then incubated with the primary antibody, mouseanti-tktl1 (1:300) (for 1 hour at RT, the slides were then rinsed withwashing buffer and placed in a fresh buffer bath for 5 min. The antibodyemployed is directed against the protein sequence shown in bold in FIG.7 of human tktl1.

Afterwards the slides were incubated with the secondary antibody (goatanti mouse (1:500)) for 1 hour at RT. Washing was performed 3 times for5 minutes. Slides were covered with 200 μl substrate-chromogen solution(DAB) for 10 min. Then slides were washed as before and counterstainedfor 2 min in a bath of haematoxylin. Residual haematoxylin was rinsedwith distilled water, and specimens were mounted and coverslipped withan aqueous mounting medium.

The microscopic examination of the slides reveals, that cellsimmunoreactive with tktl1 can be found in samples, that maymicroscopically be identified as samples of gastric carcinoma. Incarcinomas the tktl1 specific staining is visible in the nucleus and thecytoplasm. In addition a granular staining pattern was observed in tumorcells.

The above described immunohistochemical staining procedure wasfurthermore applied to tissues from breast-, lung-, cervical- (CINIII),gastric-, oesophageal-, endometrial-, ovarian-carcinomas. In all thesecases nuclear and cytoplasmic staining for tktl1 could be observed inthe cancerous cells.

Moreover metastases from colorectal carcinoma located in the liver wereanalysed by immunochemical procedures as described above. The resultshowed a strong overexpression of the tktl1 protein.

1. An in vitro method for detection of carcinoma in an individualcomprising: (a) obtaining a suspected carcinoma sample from anindividual; (b) detecting in the sample obtained from said individualthe level of transketolase like-1 polypeptide encoded by transketolaselike-1 gene; (c) comparing the level detected in step (b) with the levelof transketolase like-1 polypeptide encoded by a transketolase like-1gene in a corresponding control non-carcinoma sample from a healthysubject; and (d) in the case that the sample from the individual has ahigher level of transketolase like-1 polypeptide encoded by atransketolase like-1 gene than the control tissue sample, diagnosingsaid sample as indicative of a carcinoma.
 2. The method according toclaim 1, wherein the carcinoma is colon cancer, lung cancer, gastriccancer, pancreatic cancer, cervical cancer, breast cancer, bladdercancer.
 3. The method according to claim 1, wherein in step (b) thedetection is carried out by using an antibody directed againsttransketolase like-1 protein.
 4. The method according to claim 1,wherein in step (b) the detection is carried out by using a detectablylabeled protein detecting probe.
 5. The method according to claim 4,wherein the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and a enzyme suitable to detectproteins.
 6. The method according to claim 1, wherein step (b) comprisesperforming in vitro molecular imaging.
 7. The method according to claim1, wherein said transketolase-like1 gene is as given in NCBI AccessionNo. X
 91817. 8. An in vitro method for detection of carcinoma in anindividual comprising: (a) obtaining a suspected carcinoma sample froman individual; (b) detecting in the sample obtained from said individualthe level of polypeptide encoded by polynucleotide having the nucleicacid sequence of SEQ ID NO:1; (c) comparing the level detected in step(b) with the level of polypeptide encoded by polynucleotide having thenucleic acid sequence of SEQ ID NO:1 in a corresponding controlnon-carcinoma sample from a healthy subject; and (d) in the case thatthe sample from the individual has a higher level of polypeptide encodedby polynucleotides having the nucleic acid sequence of SEQ ID NO:1 thanthe control tissue sample, diagnosing said sample as indicative of acarcinoma.
 9. The method according to claim 8, wherein the carcinoma iscolon cancer, lung cancer, gastric cancer, pancreatic cancer, cervicalcancer, breast cancer, bladder cancer.
 10. The method according to claim8, wherein in step (b) the detection is carried out by using an antibodydirected against transketolase like-1 protein.
 11. The method accordingto claim 8, wherein in step (b) the detection is carried out by using adetectably labeled protein detecting probe.
 12. The method according toclaim 11, wherein the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and a enzyme suitable to detectproteins.
 13. The method according to claim 8, wherein step (b)comprises performing in vitro molecular imaging.
 14. An in vitro methodfor detection of carcinoma in an individual comprising: (a) obtaining abiological tissue sample suspected to contain cancerous cells from anindividual; (b) detecting in said biological tissue sample obtained fromsaid individual the level of transketolase like-1 polypeptide encoded bytransketolase like-1 gene; (c) comparing the results of step (b) with areference value obtained by detecting, in a normal control sample of thesame type as the suspected cancerous biological tissue sample but knownto be non-cancerous, the level of transketolase like-1 polypeptideencoded by a transketolase like-1 gene; and (d) in the case that ahigher level of transketolase like-1 polypeptide is detected in saidsuspected cancerous biological tissue sample suspected to containcancerous cells as compared to said level of transketolase like-1polypeptide in said normal control sample, diagnosing said individual ashaving a carcinoma.
 15. The method according to claim 14, wherein thecarcinoma is colon cancer, lung cancer, gastric cancer, pancreaticcancer, cervical cancer, breast cancer, bladder cancer.
 16. The methodaccording to claim 14, wherein in step (b) the detection is carried outby using an antibody directed against transketolase like-1 protein. 17.The method according to claim 14, wherein in step (b) the detection iscarried out by using a detectably labeled protein detecting probe. 18.The method according to claim 17, wherein the label is selected from thegroup consisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, anda enzyme suitable to detect proteins.
 19. The method according to claim1, wherein step (b) comprises performing in vitro molecular imaging. 20.The method according to claim 1, wherein said transketolase-like1 geneis as given in NCBI Accession No. X
 91817. 21. An in vitro method fordetection of carcinoma in an individual comprising: (a) obtaining abiological tissue sample suspected to contain cancerous cells from anindividual; (b) detecting in said biological tissue sample obtained fromsaid individual the level of polypeptide comprising the amino acidsequence encoded by polynucleotides having the nucleic acid sequence ofSEQ ID NO:1; (c) comparing the results of step (b) with a referencevalue obtained by detecting, in a normal control sample of the same typeas the suspected cancerous biological tissue sample but known to benon-cancerous, the level of polypeptide comprising the amino acidsequence encoded by polynucleotides having the nucleic acid sequence ofSEQ ID NO:1; and (d) in the case that a higher level of polypeptides isdetected in said suspected cancerous biological tissue sample suspectedto contain cancerous cells as compared to said level of polypeptides insaid normal control sample, diagnosing said individual as having acarcinoma.
 22. The method according to claim 21, wherein the carcinomais colon cancer, lung cancer, gastric cancer, pancreatic cancer,cervical cancer, breast cancer, bladder cancer.
 23. The method accordingto claim 21, wherein in step (b) the detection is carried out by usingan antibody directed against transketolase like-1 protein.
 24. Themethod according to claim 21, wherein in step (b) the detection iscarried out by using a detectably labeled protein detecting probe. 25.The method according to claim 24, wherein the label is selected from thegroup consisting of a radioisotope, a bioluminescent compound, achemiluminescent compound, a fluorescent compound, a metal chelate, anda enzyme suitable to detect proteins.
 26. The method according to claim21, wherein step (b) comprises performing in vitro molecular imaging.27. An in vitro method for detection of carcinoma in an individualcomprising: (a) obtaining a biological test sample from an individualsuspected to be suffering of carcinoma, said test sample being selectedfrom the group consisting of serum, blood, plasma, urine, semen, stool,bile, a biopsy or a cell- or tissue-sample, gastric juice, andpancreatic juice; (b) detecting in said biological test sample obtainedfrom said individual the level of transketolase like-1 polypeptideencoded by transketolase like-1 gene; (c) comparing the results of step(b) with a reference value obtained by detecting, in a normal controlsample of the same type and, in the case of tissue, of the same tissuetype, as the test sample but known to be non-cancerous, the level oftransketolase like 1 polypeptide encoded by the transketolase like-1gene; and (d) in the case that a higher level of transketolase like 1polypeptide is detected in said biological test sample as compared tosaid level of transketolase like 1 polypeptide in said normal controlsample, diagnosing said individual as having a carcinoma.
 28. The methodaccording to claim 27, wherein the carcinoma is colon cancer, lungcancer, gastric cancer, pancreatic cancer, cervical cancer, breastcancer, bladder cancer.
 29. The method according to claim 27, wherein instep (b) the detection is carried out by using an antibody directedagainst transketolase like-1 protein.
 30. The method according to claim27, wherein in step (b) the detection is carried out by using adetectably labeled protein detecting probe.
 31. The method according toclaim 30, wherein the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and a enzyme suitable to detectproteins.
 32. The method according to claim 27, wherein step (b)comprises performing in vitro molecular imaging.
 33. The methodaccording to claim 27, wherein said transketolase-like1 gene is as givenin NCBI Accession No. X
 91817. 34. An in vitro method for detection ofcarcinoma in an individual comprising: (a) obtaining a biological testsample from an individual suspected to be suffering of carcinoma, saidtest sample being selected from the group consisting of serum, blood,plasma, urine, semen, stool, bile, a biopsy or a cell- or tissue-sample,gastric juice, and pancreatic juice; (b) detecting in said biologicaltest sample obtained from said individual the level of polypeptideencoded by polynucleotides having the nucleic acid sequence of SEQ IDNO:1; (c) comparing the results of step (b) with a reference valueobtained by detecting, in a normal control sample of the same type and,in the case of tissue, of the same tissue type, as the test sample butknown to be non-cancerous, the level of polypeptide encoded bypolynucleotides having the nucleic acid sequence of SEQ ID NO:1; and (d)in the case that a higher level of polypeptides is detected in saidbiological test sample as compared to said level of polypeptides in saidnormal control sample, diagnosing said individual as having a carcinoma.35. The method according to claim 34, wherein the carcinoma is coloncancer, lung cancer, gastric cancer, pancreatic cancer, cervical cancer,breast cancer, bladder cancer.
 36. The method according to claim 34,wherein in step (b) the detection is carried out by using an antibodydirected against transketolase like-1 protein.
 37. The method accordingto claim 34, wherein in step (b) the detection is carried out by using adetectably labeled protein detecting probe.
 38. The method according toclaim 37, wherein the label is selected from the group consisting of aradioisotope, a bioluminescent compound, a chemiluminescent compound, afluorescent compound, a metal chelate, and a enzyme suitable to detectproteins.
 39. The method according to claim 34, wherein step (b)comprises performing in vitro molecular imaging.